Association of terminal deoxynucleotidyl transferase with Ku.

Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity. Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair. We provide evidence for a direct interaction between TdT and Ku proteins. Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer. The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins. The protein-protein interaction is DNA independent and occurs at physiological salt concentrations. Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer. This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair. The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize. The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.

T-cell specific avian TdT: characterization of the cDNA and recombinant enzyme.

A cDNA clone coding for avian terminal deoxynucleotidyl transferase (TdT) has been isolated and sequenced. The size of this cDNA was 2545 bp with an open reading frame of 1521 bp and a predicted translation product of 58 kDa. Comparison of this TdT sequence with other known TdT sequences has revealed a very high degree of homology at both the DNA and predicted amino acid levels. The chicken TdT cDNA was expressed in a bacterial system and the protein was purified by affinity chromatography. The purified recombinant enzyme, with a specific activity of approximately 1700 U/mg protein, was significantly less active than TdTs from mammalian species. This finding correlates with the observation that TdT isolated from avian thymus has lower activity than that isolated from any mammalian thymus source. Northern blot hybridization analyses and reverse transcription PCR of RNA preparations were carried out with the chicken cDNA. The data generated from these experiments revealed that the TdT RNA was only expressed in the thymus and not in the bone marrow or the bursa of Fabricius during pre- and post hatching chicken development. These data suggest that while TdT is probably involved in N region addition in chicken T-cell receptor genes, it is unlikely to play a role in diversification of immunoglobulin genes.

Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase.

Human terminal deoxynucleotidyl transferase (TdT) was overexpressed in a baculovirus system. The pure recombinant enzyme was identical in size, activity, kinetic constants, and metal effects to native enzyme. Three amino acids, within either the putative nucleotide binding domain and part of a DNA polymerase consensus sequence, YGDTDSLF, or a TdT consensus sequence, GGFRRGK, were altered by site-directed mutagenesis. The four mutant forms of terminal transferase were also overexpressed in the baculovirus expression system and purified from Trichoplusia ni larvae by a monoclonal antibody affinity column and compared with wild-type enzyme with respect to thermostabilities, secondary structure, metal effects, and kinetic parameters. Three of the four mutants retained 3-16% of wild-type activity under varying metal conditions, and one of the mutants, D343E, consistently exhibited less than 0.2% of wild-type TdT activity with dATP and no activity with dGTP. All mutants had alterations in the Km for dATP. Variations in Km for dGTP were not as consistent. The Km for the other substrate, DNA initiator (dA)50) in the presence of dATP remained essentially the same as that of wild-type TdT for all mutants except D343E. The enzyme activity of all mutants was stimulated by Zn2+ at low concentrations, and this effect was diminished and reversed at higher concentrations of ZnSO4. All mutants still retained significant amounts of the secondary structure as measured by circular dichroism. These results indicated that the aspartic acid residue at position 343 is located at or near the active site and is critical for the nucleotide binding and catalytic activity.